Thermo Fisher Scientific Axiom Propel Workflow, 96‑Array Format User guide

For Research Use Only. Not for use in diagnostic procedures.

Axiom™ Propel Workﬂow, 96‑Array Format

USER GUIDE

for use with:

Axiom™ Array Plates

Axiom™ Propel Reagent Kits

Multidrop™ Combi Reagent Dispenser

Catalog Numbers 952341 and 952342

Publication Number MAN0018845

Revision A.0

Manufacturer:

Thermo Fisher Scientiﬁc Baltics

UAB |

V.A. Graiciuno 8, LT-02241 |

Vilnius, Lithuania

Products:

Axiom™ Propel 4x96F Reagent Kit

Axiom™ Propel 8x96F Reagent Kit

Manufacturer:

Affymetrix Pte Ltd |

7 Gul Circle #2M-01 |

Keppel Logistics Building |

Singapore 629563

Products:

Axiom™ Array Plates

Axiom™ myDesign™ Array Plates

Thermo Fisher Scientiﬁc Oy |

Ratastie 2 | FI-01620 Vantaa |

Finland

Products:

Multidrop™ Combi Reagent Dispenser

The information in this guide is subject to change without notice.

DISCLAIMER: TO THE EXTENT ALLOWED BY LAW, THERMO FISHER SCIENTIFIC INC. AND/OR ITS AFFILIATE(S) WILL NOT BE LIABLE FOR SPECIAL,

INCIDENTAL, INDIRECT, PUNITIVE, MULTIPLE, OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT,

INCLUDING YOUR USE OF IT.

Revision history: Pub. No. MAN0018845

Revision Date Description

A.0 27 January 2019 New publication.

Important Licensing Information: These products may be covered by one or more Limited Use Label Licenses. By use of these products, you accept

the terms and conditions of all applicable Limited Use Label Licenses.

TRADEMARKS: All trademarks are the property of Thermo Fisher Scientiﬁc and its subsidiaries unless otherwise speciﬁed. UV-Star is a registered

trademark of Greiner Bio-One GmbH. BINDER is a trademark of BINDER GmbH. Sigma-Aldrich is a trademark of Sigma-Aldrich Co., LLC. Promega is

a trademark of Promega Corporation. GripTips is a trademark of Integra Biosciences Corp. minION is a trademark of Simco-Ion, Technology Group.

Signature is a trademark of VWR International, LLC. Corning is a registered trademark of Corning Incorporated.

Contents

■CHAPTER 1 Overview ................................................... 11

About the Axiom™ Propel Workﬂow ............................................... 11

About the Axiom™ Genotyping Solution ....................................... 11

Axiom™ Propel Workﬂow target preparation overview .............................. 13

Dispense, seal, shake, then centrifuge ............................................ 14

GeneTitan™ reagent tray barcodes ............................................... 15

Overview of the Axiom™ Assay workﬂow .......................................... 16

Multi-plate workﬂows ...................................................... 17

■CHAPTER 2 Genomic DNA preparation ............................... 18

Sources of genomic DNA ........................................................ 18

General requirements .......................................................... 19

Special requirements ...................................................... 19

Evaluate the quality of genomic DNA with 1% agarose E‑Gel™................... 20

Genomic DNA extraction/puriﬁcation methods ..................................... 21

Genomic DNA cleanup .......................................................... 21

Genomic DNA preparation ....................................................... 22

Genomic DNA input requirements ............................................ 22

Time required ............................................................. 22

Equipment, consumables, and reagents required .............................. 22

Thaw samples and control .................................................. 23

Quantify and dilute test sample gDNA ........................................ 24

Aliquot the diluted samples and the control ................................... 24

Freeze or proceed ......................................................... 24

GeneTitan™ Array Plate Registration ﬁle .......................................... 24

Create and save a GeneTitan™ Array Plate Registration ﬁle ...................... 25

■CHAPTER 3 Set up for the Axiom™ Propel Workﬂow,

96‑Array Format .......................................................... 26

Required materials ............................................................. 26

Equipment and materials required .......................................... 26

Labware and consumable ordering information ............................... 28

Axiom™ Propel Reagent Kit ................................................. 31

Additional reagents and materials required ................................... 33

Axiom

™

Propel Workﬂow, 96‑Array Format User Guide

3

GeneTitan™ bulk consumables .............................................. 33

Other equipment, consumables, and reagents required ......................... 36

Guidelines for handling plates and reagents ....................................... 40

Best practices for Axiom™ Propel Workﬂow, 96‑Array Format ........................ 41

Preampliﬁcation area ...................................................... 41

General guidelines ......................................................... 41

Master mix preparation guidelines ........................................... 42

Multidrop™ Combi use guidelines ............................................ 42

VIAFLO guidelines ......................................................... 43

Plate heat sealer guidelines ................................................. 43

Shaker guidelines ......................................................... 44

Centrifuge guidelines ...................................................... 44

Multidrop™ Combi Reagent Dispenser operations .................................. 45

Multidrop™ Combi protocol names and parameters ............................ 45

Materials required ......................................................... 46

Start up the Multidrop™ Combi ................................................... 47

Perform gravimetric checks ..................................................... 47

Range guidelines for gravimetric tests ....................................... 48

Prime the cassette ............................................................. 50

Flush the Multidrop™ Combi between batches of plates ............................. 50

Shut down the Multidrop™ Combi Reagent Dispenser ............................... 51

■CHAPTER 4 Target preparation with Multidrop™ Combi

Reagent Dispensers for 8 plates ........................................ 53

Stage 1: Amplify the genomic DNA ............................................... 54

Equipment and labware required ............................................ 54

Reagent handling .......................................................... 55

Prepare Denaturation Master Mix ............................................ 55

Prepare the Axiom™ Neutral Solution ........................................ 56

Prepare the Ampliﬁcation Master Mix ........................................ 56

Stage 1 summary .......................................................... 57

Perform the pre‑run checklist ............................................... 57

Stage 1: Amplify the genomic DNA ........................................... 58

Stage 2: Fragment the DNA ...................................................... 61

Equipment and labware required ............................................ 61

Reagent and plate handling ................................................. 61

Prepare the Fragmentation Master Mix ...................................... 63

Prepare the Axiom™ Frag Reaction Stop ...................................... 63

Stage 2 summary .......................................................... 64

Contents

4

Axiom

™

Propel Workﬂow, 96‑Array Format User Guide

Perform the pre‑run checklist ............................................... 64

Stage 2: Fragment the DNA ................................................. 65

Stage 3: Precipitate the DNA ..................................................... 67

Equipment and labware required ............................................ 67

Prepare Precipitation Master Mix ............................................ 67

Prepare the Isopropanol .................................................... 68

Stage 3 summary ......................................................... 68

Perform the pre‑run checklist ............................................... 68

Stage 3: Precipitate the DNA ................................................ 69

Stage 4: Centrifuge and dry DNA pellets .......................................... 70

Equipment required ........................................................ 70

Stage 4 summary .......................................................... 70

Perform the pre‑run checklist ............................................... 70

Stage 4: Centrifuge and dry pellets ........................................... 71

Stage 5: Resuspend the pelleted DNA and prepare for hybridization .................. 72

Equipment and labware required ............................................ 72

Reagent and plate handling ................................................. 73

Guidelines for pellet preparation ............................................ 73

Prepare the Axiom™ Resuspension Buffer .................................... 74

Prepare Hybridization Master Mix ............................................ 74

Stage 5 summary .......................................................... 74

Perform the pre‑run checklist ............................................... 75

Stage 5: Resuspend the pelleted DNA and prepare for hybridization .............. 76

Transfer the hybridization-ready target to the 96PCR plate ...................... 78

Stage 5A: In-process QC ........................................................ 81

Equipment and labware required ............................................ 81

Reagents required ......................................................... 81

Stage 5A summary ......................................................... 81

Stage 5A: In-process QC ................................................... 82

Transfer hybridization-ready target in 96 PCR plate to QC plate .................. 84

Stage 6: Denature the target and transfer to hybridization tray ....................... 89

Equipment and labware required ............................................ 89

Stage 6 summary .......................................................... 89

Perform the pre‑run checklist ............................................... 89

Warm the array plate to room temperature ................................... 89

Stage 6: Denature the target and transfer to hybridization tray .................. 90

Transfer hybridization-ready target in 96 PCR plate to hybridization tray .......... 91

Perform off-line hybridization ............................................... 94

Stage 7: Preparing ligation, stain, and stabilization reagent trays for the

GeneTitan™ MC Instrument ...................................................... 96

Equipment and labware required ............................................ 96

Reagent handling .......................................................... 96

Reagent preparation for Stage 7: Prepare GeneTitan™ reagents ................. 99

Prepare Ligate Master Mix .................................................. 99

Prepare Stain 1 Master Mix ................................................ 100

Contents

Axiom

™

Propel Workﬂow, 96‑Array Format User Guide

5

Prepare Stain 2 Master Mix ................................................ 101

Prepare Stabilization Master Mix ........................................... 101

Prepare the Axiom™ Hold Buffer ............................................ 102

Stage 7 summary ......................................................... 102

Prepare the GeneTitan™ trays and covers .................................... 102

Perform the pre‑run checklist .............................................. 103

Stage 7: Prepare GeneTitan™ reagents ...................................... 104

■CHAPTER 5 Target preparation with Multidrop™ Combi

Reagent Dispensers for 4 plates ....................................... 109

Stage 1: Amplify the genomic DNA .............................................. 110

Equipment and labware required ........................................... 110

Reagent handling ......................................................... 111

Prepare Denaturation Master Mix ........................................... 111

Prepare the Axiom™ Neutral Solution ....................................... 112

Prepare the Ampliﬁcation Master Mix ....................................... 112

Stage 1 summary ......................................................... 113

Perform the pre‑run checklist .............................................. 113

Stage 1: Amplify the genomic DNA .......................................... 114

Stage 2: Fragment the DNA .................................................... 117

Equipment and labware required ........................................... 117

Reagent handling ......................................................... 118

Prepare the Fragmentation Master Mix ..................................... 119

Prepare the Axiom™ Frag Reaction Stop ..................................... 119

Stage 2 summary ......................................................... 120

Perform the pre‑run checklist .............................................. 120

Stage 2: Fragment the DNA ................................................ 121

Stage 3: Precipitate the DNA ................................................... 123

Equipment and labware required ........................................... 123

Prepare Precipitation Master Mix ........................................... 123

Prepare the Isopropanol ................................................... 124

Stage 3 summary ........................................................ 124

Perform the pre‑run checklist .............................................. 124

Stage 3: Precipitate the DNA ............................................... 125

Stage 4: Centrifuge and dry DNA pellets ......................................... 126

Equipment required ....................................................... 126

Stage 4 summary ......................................................... 126

Perform the pre‑run checklist .............................................. 126

Stage 4: Centrifuge and dry pellets ......................................... 127

Stage 5: Resuspend the pelleted DNA and prepare for hybridization ................. 128

Equipment and labware required ........................................... 128

Reagent handling ......................................................... 129

Guidelines for pellet preparation ........................................... 129

Prepare the Axiom™ Resuspension Buffer ................................... 130

Prepare Hybridization Master Mix ........................................... 130

Contents

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™

Propel Workﬂow, 96‑Array Format User Guide

Stage 5 summary ......................................................... 130

Perform the pre‑run checklist .............................................. 131

Stage 5: Resuspend the pelleted DNA and prepare for hybridization ............. 132

Transfer the hybridization-ready target to the 96PCR plate ..................... 134

Stage 5A: In-process QC ....................................................... 137

Equipment and labware required ........................................... 137

Stage 5A summary ........................................................ 137

Stage 5A: In-process QC .................................................. 138

Transfer hybridization-ready target in 96 PCR plate to QC plate ................. 140

Stage 6: Denature the target and transfer to hybridization tray ...................... 145

Equipment and labware required ........................................... 145

Stage 6 summary ......................................................... 145

Perform the pre‑run checklist .............................................. 145

Warm the array plate to room temperature .................................. 145

Stage 6: Denature the target and transfer to hybridization tray ................. 146

Transfer hybridization-ready target in 96 PCR plate to hybridization tray ......... 146

Perform off-line hybridization .............................................. 149

Stage 7: Preparing ligation, stain, and stabilization reagent trays for the

GeneTitan™ MC Instrument ..................................................... 152

Equipment and labware required ........................................... 152

Reagent handling ......................................................... 152

Reagent preparation for Stage 7: Prepare GeneTitan™ reagents ................ 154

Prepare Ligate Master Mix ................................................. 155

Prepare Stain 1 Master Mix ................................................ 156

Prepare Stain 2 Master Mix ................................................ 156

Prepare Stabilization Master Mix ........................................... 157

Prepare the Axiom™ Hold Buffer ............................................ 157

Stage 7 summary ......................................................... 158

Prepare the GeneTitan™ trays and covers .................................... 158

Perform the pre‑run checklist .............................................. 159

Stage 7: Prepare GeneTitan™ reagents ...................................... 160

■CHAPTER 6 Process array plates with the GeneTitan™ Multi-

Channel Instrument .................................................... 165

Create and upload a GeneTitan™ Array Plate Registration ﬁle ....................... 165

Run Wash Scan ............................................................... 166

Continue the scan workﬂow .................................................... 174

Shut down the GeneTitan™ MC Instrument ....................................... 175

Contents

Axiom

™

Propel Workﬂow, 96‑Array Format User Guide

7

■CHAPTER 7 Scalable high throughput with the

Axiom™ Propel Workﬂow ............................................... 176

Overview ..................................................................... 176

Considerations for individualized scale-up of the Axiom™ Assay Axiom™ Propel

Workﬂow .................................................................... 177

GeneTitan™ MC Instrument throughput ...................................... 177

Target preparation with the Axiom™ Propel Workﬂow ......................... 177

32x96-array format plates per week ............................................. 178

Requirements and output .................................................. 178

Day-by-day activities ...................................................... 179

96x96-array format plates per week ............................................. 180

Requirements and output .................................................. 180

Day-by-day activities ...................................................... 181

■APPENDIX A Recommended techniques for GeneTitan™ MC

Instrument operation ................................................... 186

Array plate packaging ......................................................... 186

Proper tray alignment and placement ........................................... 187

Proper orientation of consumables ......................................... 189

Drawer ﬁngers in the GeneTitan™ MC Instrument ............................. 189

Stain trays and covers ......................................................... 191

Label GeneTitan™ hybridization and reagent trays ................................. 191

Label GeneTitan™ hybridization trays ........................................ 191

Label GeneTitan™ reagent trays ............................................ 192

Guidelines for aliquoting reagents to GeneTitan™ trays ............................ 192

Deionization of GeneTitan™ trays and covers ...................................... 194

Manual deionization of GeneTitan™ trays and covers .......................... 194

Setup options for array plate processing ......................................... 198

Hyb-Wash-Scan .......................................................... 198

Hyb-Wash ............................................................... 199

Wash-Scan ............................................................... 199

Wash-Scan Resume ....................................................... 199

Scan .................................................................... 200

Unload Plates ............................................................ 200

Wash .................................................................... 200

Load an array plate and hybridization tray into the GeneTitan™ MC Instrument

(for Hyb-Wash-Scan or Hyb-Wash) .............................................. 201

Load a second array plate and hybridization tray onto the GeneTitan™ MC Instrument . 205

When a second array plate and hybridization tray can be loaded ................ 205

Load a second array plate and hybridization tray .............................. 206

Abort a process ............................................................... 207

How to abort a process .................................................... 208

Contents

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Axiom

™

Propel Workﬂow, 96‑Array Format User Guide

Email notiﬁcations from the GeneTitan™ MC Instrument ........................... 208

GeneTitan™ MC Instrument lamp ................................................ 209

■APPENDIX B Fragmentation quality control gel protocol .......... 210

Equipment required ........................................................... 210

E‑Gel™ and reagents required .................................................. 210

Consumables required ......................................................... 211

Prepare the gel diluent ........................................................ 211

Run the fragmentation QC gel .................................................. 211

■APPENDIX C Sample quantiﬁcation after resuspension ........... 213

Equipment required ........................................................... 213

Quantify the diluted samples ................................................... 213

Install Axiom™ OD methods on the Multiskan™ Sky Microplate Spectrophotometer .... 214

Use a Multiskan™ Sky session .................................................. 220

OD yield evaluation guidelines .................................................. 220

Plate reader guidelines for sample quantiﬁcation ................................. 221

■APPENDIX D Register samples in GeneChip™ Command

Console™................................................................. 222

GeneTitan™ Array Plate Registration ﬁle ......................................... 222

Create a GeneTitan™ Array Plate Registration ﬁle ................................. 222

■APPENDIX E Troubleshooting ........................................ 225

Multidrop™ Combi Reagent Dispenser ........................................... 225

Adjust Multidrop™ Combi dispense volume ....................................... 225

Thermo Scientiﬁc™ ALPS 3000 Automated Microplate Heat Sealer .................. 226

ALPS 3000 Automated Microplate Heat Sealer ................................... 226

Align the ALPS 3000 seal roll ............................................... 227

Clean the ALPS 3000 Automated Microplate Heat Sealer ....................... 229

Park the ALPS 3000 Automated Microplate Heat Sealer ....................... 229

GeneTitan™ Instrument support ﬁles for troubleshooting .......................... 231

Log ﬁles ................................................................. 231

GeneChip™ Command Console™ log ﬁles ..................................... 231

Other GeneChip™ Command Console™ ﬁles .................................. 232

GCC log ﬁles for GeneTitan™ MC Instrument systems .......................... 232

GeneTitan™ MC Instrument ..................................................... 233

GeneTitan™ Instrument ﬂuidic diagnostic messages ............................... 235

Contents

Axiom

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Propel Workﬂow, 96‑Array Format User Guide

9

■APPENDIX F GeneTitan™ Multi-Channel Instrument care ......... 238

Overview ..................................................................... 238

Maintenance ................................................................. 238

Monthly ................................................................. 238

Every six months ......................................................... 238

Outer enclosure fan ﬁlters ..................................................... 239

Cleaning schedule ........................................................ 239

Clean the GeneTitan™ MC Instrument fan ﬁlter ............................... 239

Bottle ﬁlter replacement ....................................................... 239

Remove and inspect the reagent bottle ﬁlters ................................ 240

Replace ﬂuidics bottle ﬁlter ................................................ 241

Xenon lamp replacement in the GeneTitan™ MC Instrument ........................ 241

Lamp life/imaging device status notices ..................................... 241

Remove the xenon lamp ................................................... 242

Replace the xenon lamp ................................................... 244

Reset the lamp life counter ................................................ 245

■APPENDIX G Safety ................................................... 246

Symbols on this instrument .................................................... 246

Standard safety symbols ................................................... 246

Additional safety symbols .................................................. 248

Location of safety labels ................................................... 250

Control and connection symbols ............................................ 251

Conformity symbols ....................................................... 251

Safety information for instruments not manufactured by Thermo Fisher Scientiﬁc .... 253

Chemical safety ............................................................... 253

Biological hazard safety ........................................................ 255

■APPENDIX H Documentation and support .......................... 256

Related documentation ........................................................ 256

Customer and technical support ................................................ 258

Limited product warranty ...................................................... 258

Contents

10

Axiom

™

Propel Workﬂow, 96‑Array Format User Guide

Overview

About the Axiom™ Propel Workﬂow

The Axiom™ Propel Workow, 96-Array Format is a new workow for ultra high-

throughput microarray genotyping. The workow includes:

• DNA target preparation using Multidrop™ Combi Reagent Dispenser stations

setup for: DNA amplication, fragmentation, purication, and resuspension of

the pelleted DNA in hybridization cocktail.

• Denature the hybridization-ready target DNA and hybridize sample plates in an

o-line oven.

• Transfer to the Applied Biosystems™ GeneTitan™ Multi-Channel (MC) Instrument

followed by automated staining, washing, and imaging.

• Processing of CEL les generated by the GeneTitan™ MC Instrument, using the

Axiom™ Genotyping Algorithm version 1 (Axiom GT1), available through

Applied Biosystems™ Array Power Tools or Axiom™ Analysis Suite v2.0 or later.

The Axiom™ Propel Reagent Kit provides all necessary large-lled reagents for target

preparation and GeneTitan™ reagents in volumes that are optimized for processing

the modular workow.

IMPORTANT! The Applied Biosystems™ Axiom™ Propel Reagent Kit is for single use

only. This large ll reagent kit is congured to include prime volumes required for use

with the Multidrop™ Combi. Discard all excess reagents after use.

The Axiom™ Propel Workow, 96-Array Format is part of the Axiom™ Genotyping

Solution. The Axiom™ Genotyping Solution is a genotyping microarray platform that

includes novel assay biochemistry, array conguration and processing, and

automated target preparation on various array plate formats. It oers the capability to

genotype hundreds of thousands of single nucleotide polymorphisms (SNPs) and

insertion/deletion polymorphisms (indels) from a variety of species, with a processing

throughput of greater than 3,000 samples per week.

1

About the Axiom™

Genotyping

Solution

Axiom

™

Propel Workﬂow, 96‑Array Format User Guide

11

High-throughput genotyping through microarray technology has applications in

human disease research and basic and applied agriculture research.

• For human disease research applications, Thermo Fisher Scientic conducted an

empirical screen of genomic content from dbSNP (ncbi.nlm.nih.gov/projects/

SNP/). The screen included markers from HapMap and the 1,000 Genomes

Project and other sources, using HapMap phase 3 samples and/or the original 270

HapMap samples. All this information has gone into creating a proprietary

database of veried markers that can be interrogated using the Axiom™ Assay.

• For agriculture applications, the Axiom™ Genotyping Solution can genotype

samples using DNA extracted from leaves and seeds, playing an important role

in genotype-trait association studies and marker-assisted selection in both plant

and animal breeding programs.

• For molecular breeding programs, where turn-around time, accuracy, and ease-

of-use are all important, the Axiom™ Genotyping Solution is ideal for high-

throughput analysis.

The Axiom™ 96-array layout and the Axiom™ 384HT-array layout retain full

compatibility with the existing Axiom™ instrumentation platform and downstream

data analysis.

Chapter 1 Overview

About the Axiom

™

Propel Workﬂow

1

12

Axiom

™

Propel Workﬂow, 96‑Array Format User Guide

Axiom™ Propel Workﬂow target preparation overview

Assay stage Instruments required

Stage 1: Amplify the genomic DNA

• Three reagent additions with mixing—Denaturation Master Mix,

Axiom™ Neutral Solution, Ampliﬁcation Master Mix

• 10-minute denature incubation at room temperature

• 22–24 hour ampliﬁcation incubation at 37°C

Stage 2: Fragment the DNA

• Two reagent additions with mixing—Fragmentation Master Mix,

Axiom™ Frag Reaction Stop

• 30-minute fragmentation incubation at 37°C

Stage 3: Precipitate the DNA

• Two reagent additions with high-speed mixing—Precipitation

Master Mix and isopropanol

• Overnight precipitation

20°C

Stage 4: Centrifuge and dry pellets

Purify ampliﬁed DNA into dried pellets.

Stage 5: Resuspend the pelleted DNA and prepare for hybridization

• Two reagent additions with mixing—Axiom™ Resuspension

Buffer, Hybridization Master Mix

• 10 minute shaking to resuspend the DNA pellets

• Transfer from deep-well plate to PCR plate

Stage 5A: In-process QC

• Three reagent dispenses—Dilution Plates, OD Plates, Gel Plates

• Transfer hybridization-ready target from 96 PCR plate to QC

Plates

Stage 6: Denature the target and transfer to hybridization tray

• Denature target in thermal cycler

• Transfer from PCR plate to hybridization tray

• Off-line incubation of the array plate/hybridization tray stack at

48°C for 23.5–24 hours

Stage 7: Prepare GeneTitan™ reagents

Five reagent dispenses—Ligation, Stain 1, Stain 2, Stabilization,

Axiom™ Hold Buffer

Chapter 1 Overview

Axiom

™

Propel Workﬂow target preparation overview

1

Axiom

™

Propel Workﬂow, 96‑Array Format User Guide

13

Dispense, seal, shake, then centrifuge

For each stage of the Axiom™ Propel Workow, 96-Array Format conducted at a

Multidrop™ Combi Reagent Dispenser, the following steps are typically performed.

• Task name is provided in the table heading.

• Number of plates for the workow is listed in the rst row of the table.

• Each subsequent row in the table lists a step in the task/procedure, with specic

details listed.

Multidrop™ Combi tasks

Plates 1—4

1

Dispense

• Method: 96-xyz

• Dispense volume: ## μL

2

Seal

• Settings: 164°C, 2.2 seconds

3

Shake

• Settings: 1,100 rpm, 30 seconds

4

Centrifuge

• Centrifuge at room temperature

• Settings: 675 ×

g

, 30 second

Chapter 1 Overview

Dispense, seal, shake, then centrifuge

1

14

Axiom

™

Propel Workﬂow, 96‑Array Format User Guide

GeneTitan™ reagent tray barcodes

GeneTitan™ MC Instrument consumables and Applied Biosystems™ GeneChip™

Command Console™ (GCC) are required for the preparation of the Axiom™ stain

reagents. Each tray has a unique part number and barcode that oers traceability.

These trays have the following labels and barcodes:

1

2

3

4

FOR RESEARCH

USE ONLY

FOR RESEARCH

USE ONLY

FOR RESEARCH

USE ONLY

FOR RESEARCH

USE ONLY

96 Layout GeneTitan

5010251234567070914587

Stain Tray

TM

96 Layout Axiom

50139541234567070914598

Stain2 Tray

TM

96 Layout Axiom

5013991234567070914606

Ligation Tray

TM

96 Layout Axiom

5013971234567070914599

Stabilization Tray

TM

Figure 1 GeneTitan™ reagent tray barcodes and color-coded labels

1Stain 1 Tray—Part No. 501279

2Stain 2 Tray—Part No. 501394

3Ligation Tray—Part No. 501398

4Stabilization Tray—Part No. 501396

The unique barcodes along with the GeneChip™ Command Console™ v3 or later

software prevents users from making errors when placing the trays in the GeneTitan™

MC Instrument during array processing.

After the trays have been prepared, ensure that the trays are placed in the appropriate

drawer location in the GeneTitan™ MC Instrument. Failure to place the proper tray in

the correct location results in an error and the GeneTitan™ MC Instrument will not

proceed with the processing of the trays. See “Proper tray alignment and

placement“ on page 187 for detailed instruction.

GeneChip™ Command Console™ v4.3 or later also oers the facility for queuing a

second plate for scanning before the rst scan is complete. The software automatically

moves the second plate into the scanner when the rst plate has completed scanning.

Chapter 1 Overview

GeneTitan

™

reagent tray barcodes

1

Axiom

™

Propel Workﬂow, 96‑Array Format User Guide

15

Overview of the Axiom™ Assay workﬂow

Genomic DNA preparation

Day 1 Chapter 2, Genomic DNA preparation

▼

Chapter 4, “Target preparation with Multidrop™ Combi Reagent Dispensers for 8 plates“

Day 1 Stage 1: Amplify the genomic DNA 22–24 hour of Ampliﬁcation Plate at 37°C.

Optional stopping point.

The post-ampliﬁcation plates can be stored at –20°C

for up to 1 week.

▼

Day 2 Stage 2: Fragment the DNA

▼

Day 2 Stage 3: Precipitate the DNA Overnight precipitation at −20°C.

▼

Day 3 Stage 4: Centrifuge and dry DNA pellets

Optional stopping point.

The pellets can be stored at –20°C for one day.

▼

Day 3 Stage 5: Resuspend the pelleted DNA and

prepare for hybridization Optional stopping point.

The hybridization-ready target can be stored at −20°C

for up to 2 weeks.

▼

Day 3 Stage 6: Denature the target and transfer to

hybridization tray

23.5 to 24-hour array hybridization in the ofﬂine

hybridization oven at 48°C.

Day 4 Stage 7: Preparing ligation, stain, and

stabilization reagent trays for the GeneTitan™

MC Instrument

▼

Array processing

Day 4 Chapter 6, Process array plates with the

GeneTitan™ Multi-Channel Instrument

Array processing is completed with the

GeneTitan

™

MC Instrument and GeneChip

™

Command Console

™

software v6.1 or later.

Fluidics: 5.5 hours

Scan: ~5.5 hours

Chapter 1 Overview

Overview of the Axiom

™

Assay workﬂow

1

16

Axiom

™

Propel Workﬂow, 96‑Array Format User Guide

Thermo Fisher Scientic supports high-throughput workows that allow you to run a

set of samples and array plates through the protocol by using a minimum number of

personnel or exible work schedule. The timing of steps is critical because of the

following limits:

• Incubation for DNA amplication is 22–24 hours.

• Reagent trays for wash/stain/imaging must be prepared as hybridization nishes.

Contact your local support representative for more information.

Multi-plate

workﬂows

Chapter 1 Overview

Overview of the Axiom

™

Assay workﬂow

1

Axiom

™

Propel Workﬂow, 96‑Array Format User Guide

17

Genomic DNA preparation

The general requirements for genomic DNA (gDNA) sources and extraction methods

are described in this chapter. The success of this assay requires uniform amplication

of the genome starting with relatively intact gDNA. To achieve uniform amplication,

the gDNA must be of high quality, and must be free of contaminants that can aect

the enzymatic reactions to be performed.

Sources of genomic DNA

The following sources of gDNA have been successfully tested in the laboratories at

Thermo Fisher Scientic for DNA that meets the requirements for the Axiom™ Assay.

Source Sample type

Human • Blood

• Saliva

• Cell line

Animal[1] • Blood

• Semen

• Nasal swabs

• Hair bulbs

• Ear punch tissue

Plant • Seeds

• Leaves

[1] Success with sample types other than human depend on quality (degree of degradation, level of purity, and so

on) and quantity of gDNA extracted.

Note: DNA derived from formalin-xed paran-embedded (FFPE) blocks must not

be used with this assay.

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Propel Workﬂow, 96‑Array Format User Guide

General requirements

• Starting DNA must be double-stranded for accurate concentration determination.

• DNA must be of high purity. DNA must be free of DNA polymerase inhibitors.

Examples of inhibitors include high concentrations of heme (from blood) and

high concentrations of chelating agents (that is, EDTA). The gDNA extraction/

purication method must create DNA that is salt-free because high

concentrations of particular salts can also inhibit enzyme reactions. DNA purity

indicated by OD260/OD280 and OD260/OD230 ratios. The OD260/OD280 ratio should

be between 1.8 and 2.0 and the OD260/OD230 ratio should be greater than 1.5. We

recommend that DNA samples that do not meet these criteria be cleaned up as

described in “Genomic DNA cleanup“ on page 21.

• DNA must not be degraded. The average size of gDNA can be evaluated on a 1%

agarose gel using an appropriate size standard control. Approximately 90% of the

DNA must be greater than 10 Kb in size. Control DNA can be run on the same gel

for comparison.

Preampliﬁcation area

Precautions are required when manipulating genomic DNA to avoid contamination

with foreign DNA amplied in other reactions and procedures. It is recommended

that genomic DNA manipulations are performed in a dedicated preamplication

room or area separate from the main laboratory.

This preamplication area requires a dedicated set of pipees and plasticware. If no

dedicated area is available, use of a dedicated bench or a dedicated biosafety hood

and dedicated pipees is suggested. If no dedicated bench or biosafety hood is

available, a set of dedicated pipees is recommended.

Special

requirements

Chapter 2 Genomic DNA preparation

General requirements

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Propel Workﬂow, 96‑Array Format User Guide

19

We recommend this quality control step to evaluate the quality of the gDNA before

starting the assay.

Equipment and reagents required

Item Source

Mother E-Base™ Device EBM03

Daughter E-Base™ Device (optional for running multiple gels in parallel) EBD03

E‑Gel™

48

Agarose Gels, 1% G800801

Redi

Load™750026

E‑Gel™ 96 High Range DNA Marker 12352019

Guidelines for genomic DNA plate preparation

The following guidelines are recommended when preparing the genomic DNA plate

for gel analysis.

• Loading a DNA mass of 10 ng to 20 ng per well is recommended. If lower

amounts are loaded, omission of the loading dye is recommended to improve

visualization. Loading ≥25-ng gDNA per well can improve the image.

• Add 3 µL of 0.1X of RediLoad™ (RediLoad™ dye diluted 10-fold with nuclease-free

water) dye to each sample.

• Bring each sample to a total volume of 20 µL using nuclease-free water. For

example, if the volume of genomic DNA is 5 µL, add 3 µL of RediLoad™, and

bring to 20 µL total by adding 12 µL of water.

• Seal, vortex, and centrifuge briey.

Run a 48 lane 1% agarose E‑Gel™

1. Power on the E-Base™ Device (red light).

2. Push the Power/Prg buon to ensure that the gel base is in EG mode (not EP).

3. Insert the E-Gel™ 48 Agarose Gels, 1% into the slot.

4. Remove 2 combs.

5. Load 20 µL of gDNA samples onto the E-Gel™ 48 Agarose Gels, 1%.

6. Load 15 µL of diluted E-Gel™ 96 High Range DNA Marker (1:3 dilution or

~0.34 X from stock) into all marker wells (if needed).

7. Fill all empty wells with water.

8. Adjust the run time to ~27 minutes.

9. Push the Power/Prg buon again (it changes from red to green).

When run time is reached (the ladder band reaches the end of the lane), the system

automatically shuts o. The gel is ready for imaging.

Evaluate the

quality of genomic

DNA with 1%

agarose E‑Gel™

Chapter 2 Genomic DNA preparation

General requirements

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Propel Workﬂow, 96‑Array Format User Guide

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Thermo Fisher Scientific Axiom Propel Workflow, 96‑Array Format User guide

Thermo Fisher Scientific Axiom Propel Workflow, 96‑Array Format User guide

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